Mechanisms of Tolerance To Salinity in Banana: Physiological, Biochemical, and Molecular Aspects
Mechanisms of Tolerance To Salinity in Banana: Physiological, Biochemical, and Molecular Aspects
Mechanisms of Tolerance To Salinity in Banana: Physiological, Biochemical, and Molecular Aspects
ABSTRACT - In the northeastern region of Brazil, saline soils are constraints to banana production,
becoming necessary to understand the mechanisms of salt tolerance. Two bananas genotypes, Tap Maeo,
tolerant, and Berlin, sensitive, were subjected to treatment with 50 mol m-3 NaCl or without salt. This study
evaluated the effects of salt on the following physiological aspects: leaf area, content and distribution of
Na+, membrane integrity, proton ATPase activity. Besides, a search for differentially expressed genes was
performed using the Differential Display technique. Tap Maeo genotype showed the smallest reduction in
leaf area, smaller accumulation of Na+ and malondialdehyde (MDA), and higher activity of proton ATPase
activity. Two sequences differentially expressed in the tolerant genotype, (Musa 07, Musa 23), shared a
high degree of identity with the amino acid sequences of the genes SOS1 and SOS2, respectively. The clone
Musa 10 was highly similar to amino acid sequence of the ascorbate peroxidase gene, and Musa 26, encodes
the enzyme betaine aldehyde dehydrogenase. These significant biological markers indicate that salinity
tolerance in banana involves at least two simultaneous mechanisms: the activation of the SOS pathway,
increasing the extrusion of Na+, and the activation of antioxidative system, increasing the synthesis of APX
and betaine aldehyde dehydrogenase enzyme.
Index terms: DDRT-PCR, leaf area, Musa, Na+, antioxidative system, SOS pathway.
lipid peroxidation and the H+ATPase activity in the edu/tgi/cgi-bin/tgi/Blast/index.cgi), and the DNA
membrane. After fifteen days of treatment, the leaf Data Bank of Japan (DDBJ; http://www.ddbj.nig.
area was measured, and five plants were collected ac.jp/search/blast). Multiple sequence alignments
to analyze the Na+ and K+ concentrations. were performed using ClustalW software (http://
The experiment was carried out in a www.ebi.ac.uk) (THOMPSON et al., 1994), and
completely randomized design with two levels of a phylogenetic tree was constructed using the
salt (0 and 50 mol.m-3 NaCl), two cultivars (Tap neighbor-joining method with MEGA 3.1 software
Maeo and Berlin) making a total of 4 subplots. Each (KUMAR et al., 2004). The reliability of the tree
subplot contained 10 plants of each genotype, with was assessed by bootstrap analysis with 1500
a total of 40 plants. The experiment was repeated simulations and edited using MEGA 3.1 software.
3 times. The data set was submitted to analysis
of variance (ANOVA) and mean values were
compared using Tukey’s test (P < 0.05).
RESULTS AND DISCUSSION
The leaf area (LA) was determined using
a leaf area meter (Li-Cor, Inc., Lincoln, Nebraska, The salinity reduced the leaf area of both
USA). The analysis of the Na+ and K+ concentrations genotypes by different magnitudes (Table 1). In
was performed using flame photometry. the tolerant genotype, Tap Maeo, the reduction in
The plasma membrane vesicles were isolated leaf area was 21.8%, whereas the reduction in the
by differential centrifugation of the root primordial. sensitive genotype Berlin was 45.2%.
The ATPase activity of the plasma membrane was The leaf area reduction that occurs due to the
determined by measuring the Pi liberated during increase of the levels of salt is a common response
ATP hydrolysis, according to the colorimetric in banana and has been previously described in
method described by Fiske and Subbarrow, with 46 genotypes evaluated by different researchers
the modifications (FAÇANHA; DE MÉIS, 1998). (GOMES et al., 2004; SILVA et al., 2009; FERRAZ,
Vanadate (membrane ATPase inhibitor) and nitrate 2008; WILLADINO et al., 2011); these genotypes
(V-ATPase inhibitor) were used as inhibitors. exhibited reductions in leaf area that varied from
The total RNA was extracted from the roots 16.3 to 69.2%. Decreased leaf area is related to
using TRIzol. For DDRT-PCR, a 1 µg aliquot of constrained CO2 fixation, which reduces the net
total RNA was used as a standard to obtain cDNA assimilation rate and increases the production of
and for the subsequent PCR amplification. The ROS through the Mehler reaction (MILLER et
cDNA synthesis reactions were performed at 50°C al., 2009). The reduction in leaf area is also due to
for 50 minutes. The PCR reactions were performed the deviation of energy required for the activation
using combinations of anchor primers (A1, A2, A3, and maintenance of metabolic activities that are
or A4) with random primers (B1, B2, or B3). The associated with salinity tolerance, such as the
amplification conditions were as follows: 94°C for maintenance of membrane integrity, the synthesis
2 minutes, followed by 40 cycles at 94°C for 30 of organic solutes, ion extrusion, and the regulation
seconds, 42°C for 1 minute, and 72°C for 1 minute, of ion transport and distribution in various organs
as well as a final elongation stage at 72°C for 5 and within cells (MUNNS; TESTER, 2010).
minutes. PCR was performed at least three times for The K+ concentration in the shoots and roots
each primer pair. The PCR-amplified products were varied with the addition of NaCl to the nutrient
separated on 1.5% agarose gels. The genes induced solution. Whereas the sodium concentration
by salt were defined when the expression was 1.5 increased in the roots with increasing salinity but did
fold greater than that of the control, as quantified by not differ between the genotypes. In addition in the
the PCR product. The nucleotide sequences of each shoots, while the Tap Maeo genotype demonstrated
clone were determined using an automatic DNA a small increase in Na+ concentration the Berlin,
sequencer (ABI PRISM 377; Applied Biosystems, sensitive genotype, exhibited an increase of more
Foster City, California, USA), using the Big Dye than 300% (Table 1). This Na+ distribution pattern,
Terminator kit (Applied Biosystems). The BlastX which is characterized by low concentrations of
program (ALTSCHUL et al. 1997) was used to that ion in the shoots is characteristic of salinity
compare the obtained sequences with homologous tolerance, as observed in 30 banana genotypes
sequences in the databases of the National Center exposed to the same conditions (GOMES et al.,
for Biotechnology Information (NCBI; http://www. 2004; GOMES et al., 2005; WILLADINO et al.,
ncbi.nlm.nih.gov/blast), the Institute for Genomic 2011). The maintenance of ionic homeostasis in
Research (TIGR; http://compbio.dfci.harvard. the tolerant genotypes requires the extrusion of Na+
from the cytosol to the vacuole or apoplast through shoot cells of the tolerant genotype Tap Maeo
the Na+/H+ antiporter (SHI et al., 2002). (Table 1), thereby protecting the photosynthetic
The analysis of total RNA revealed apparatus. The cDNA clone Musa 23, which is
fragments corresponding to rRNA 28S and 18S differentially expressed in the Tap Maeo genotype,
with optical quality (A260/230) and density (A260/280) exhibited 73% similarity to the amino acid
ratios of approximately 1.9 and 1.7, suggesting low sequence of the SOS2 gene in Arabidopsis (access
contamination with polysaccharides and proteins, number AF237670.1) (Table 2). Greater expression
respectively, and a high yield of total RNA. of SOS2 is responsible for better salinity tolerance
Twelve primer combinations were used. response in different genotypes under salt stress
The optimal pattern and resolution clarity of the (CHAKRABORTY et al., 2012). The SOS2 gene
amplified products were obtained with the A2B2 encodes a serine/threonine protein kinase with
primer pair 48 hours after stress. either a regulatory or second catalytic domain, with
The transcripts of all of the samples (T0, T1, a regulatory-catalytic interaction within the SOS2
and T2) generated by the same pair of primers were gene. The protein kinase SOS2 interacts with a
applied side-by-side. Twenty-three differentially Ca2+-binding protein similar to calcineurin, SOS3,
expressed amplicons were isolated, cloned, and which responds to an increased Ca2+ concentration
sequenced. The length of the amplicons varied in the cytosol and triggers the interaction between
between 300 and 1900 base pairs (bp). The SOS2 and SOS3 (FEKI et al., 2014).
similarity between the sequences obtained here and The SOS2/SOS3 complex activated by sa-
previously known gene sequences was determined. line stress is responsible for the phosphorylation
Similarity was considered to be present when the and activation of SOS1, the Na+/H+ antiporter,
E values were less than (1e-05), which would which is present in the membrane (MILLER et al.,
therefore be considered significant. 2009). Therefore, the SOS2 and SOS3 proteins are
The tolerant variety, Tap Maeo, have the components of the SOS pathway. The SOS path-
differentially expressed Musa 07 cDNA fragment way ensures the ionic homeostasis of the Tap Maeo
(Table 2) in the roots that have a high homology to genotype through the extrusion of Na+ from the
SOS1 gene that encoding SOS1, the Na+/H+ antiport. cytoplasm to the apoplast or vacuole. The Na+/H+
This cDNA fragment (approximately antiporter is a secondary transporter that depends
500 bp) have an amino acid sequence with 85% on the activity of ATPases the primary transporters.
homology to the Salt Overly Sensitive 1 (SOS1) ATPases are responsible for the electrochemical
gene of the glycophyte Arabidopsis thaliana (access gradient that ensures the energization of the Na+/H+
number, AF256224) (SHI et al., 2000) (Figure 1) antiporter (VITART et al., 2004). The plants of the
and 81% homology to Pisum sativum, Thellungiella Tap Maeo genotype exhibited an increase of more
halophile, and Brassica napus. than 20% in the activity of ATPase and PPase, en-
The salt overly sensitive (SOS) pathway suring sodium extrusion, and the sensitive genotype
functionally consists of the Na+ transporter, SOS1, demonstrated a decrease in the activity of these pro-
the protein kinase SOS2, and the Ca2+ sensor SOS3, ton pumps (Figure 3).These significant biological
which constitute a functional model that ensures markers indicate that activation of the SOS pathway
cellular homeostasis in plants that are adapted or is a fundamental mechanism for saline stress toler-
tolerant to saline stress (FEKI et al., 2014; KUMAR ance of banana.
et al., 2009). The phylogenetic tree of the Na+/H+ The cDNA fragment Musa 26 encodes be-
antiport sequences (SOS1), based on higher plants, taine aldehyde protein dehydrogenase (BADH, EC
animals, fungi, and Escherichia coli, indicated 1.2.1.8), enzyme that catalyzes the oxidation of be-
two distinct “clusters”, as supported by the high taine aldehyde to glycine betaine (GB). GB plays
bootstrap value, i.e., a vacuole cluster and a plasma important role in stress tolerance that include the
membrane cluster, which indicates that Musa 07 is stabilization of complex proteins and membranes
a fragment of the Na+/H+ antiporter gene (Figure 2). in vivo, protection of the transcriptional and trans-
This same antiporter is also involved in the lational machinery, reduction of ROS accumula-
distribution of Na+ between the shoots and the root tion and peroxidation of membrane lipids, and as
(PARDO, 2010) by restricting xylem loading with a molecular chaperone in the refolding of enzymes
the Na+ ion, thereby preventing its transportation (GIRI, 2011; CHEN; MURATA, 2011). The per-
to the shoots (SUN et al., 2010; SHI et al., 2002). oxidation of membrane lipids was monitored indi-
This altered distribution of Na+ prevents toxic rectly through the production of malondialdehyde
concentrations of this cation in the cytoplasm of (MDA), a byproduct of lipid peroxidation. The Tap
Maeo genotype did not exhibit variations in the production of H2O2 and other ROS (O2-, OH-, and
MDA concentration when subjected to the stress, 1
O2) is cytotoxic (FOYER; SHIGEOKA, 2011)
however, in the Berlin genotype, the accumulation and frequently occurs in plants subjected to bi-
of Na+ caused an increase in the concentration of otic or abiotic stress (MITTLER, 2006). Tolerant
MDA (Figure 3). genotypes respond to such stress with an increase
The ability of tolerant varieties to maintain in the activity of antioxidant enzymes, preventing
low concentrations of MDA, as well as MDA ac- and avoiding damage to nucleic acids, proteins and
cumulation in susceptible varieties, indicates that membrane lipid peroxidation, thus maintaining re-
variations in lipid peroxidation of membrane can dox homeostasis (AZOOZ et al., 2009). The differ-
be used to evaluate the effect of salt stress on cell, ences on the biochemistry and molecular markers
once the membrane is the first site to perceive this highlights the importance of the antioxidant system
stress. Another cDNA fragment, the Musa 10 clone, as a mechanism of salt stress tolerance in banana.
was identified and exhibited 71% similarity to the In summary, salinity tolerance in Musa in-
amino acid sequence of the ascorbate peroxidase volves a set of at least two simultaneous mecha-
(APX) gene of Vigna unguiculata (access number nisms. These mechanisms include the activation of
U61379.1). APX is the most important enzyme as- the SOS system, which ensures the extrusion of Na+
sociated with the elimination of H2O2 from the cy- from the cytoplasm and the activation of the anti-
tosol and chloroplasts (BARBOSA et al., 2014), oxidative system, in particular the increase in the
preventing the oxidative damage. The excessive synthesis of the enzyme APX and glycine betaine.
Table 1 - Leaf area (LA), Na+ and K+ concentrations (g.kg-1), and K+/Na+ ratio in shoots (S) and roots
(R) of banana genotypes grown for 15 days with or without 50 mol m-3 NaCl in the nutrient
solution
Treatment LA Na+ K+ K+/Na+
Genotype
(mol m-3 NaCl) (cm2) S R S R S R
0 3.26 a 3.7 c 4.2 b 31.2 a 41.4 a 8.4 9.8
Tap Maeo
50 2.53 b 5.9 b 6.2 a 29.7 a 40.9 a 5.0 6.6
0 2.79 b 3.4 c 3.8 b 32.3 a 40.6 a 9.5 10.7
Berlin
50 1.26 c 16.5 a 6.9 a 30.0 a 39.9 a 1.8 5.8
LA Reduction Na+ Increase Reduction (%)
(%) (%) K+ K+/Na+
Tap Maeo 21.8 59.5 47.2 4.6 0.2 49.5 32.7
Berlin 45.2 385 81.6 7.1 1.7 89.5 45.8
Data indicated by different letters within each column exhibit significant difference at the 5 % probability level according to Tukey’s test
Figure 1 - Comparison of amino acid sequence between Musa 07 clone of Musa sp. with the SOS1 gene of
A. thaliana (AF256224). Sequences labeled with colons, asterisks, periods, and dashes represent those with
similar amino acids, identical amino acids, different amino acids, and a lack of amino acids, respectively.
The ClustalW program was used to generate the alignment (THOMPSON et al., 1997).
Figure 2 - Musa 07 clone position in the phylogenic tree of Na+/H+ antiporter. Multiple alignments of the sequences
were performed with CLUSTALW, and a phylogenic tree was built using the neighbor-joining method with the MEGA
3.1 program. The accession numbers (in parentheses) and the Na+/H+ antiporter sources are as follows: AgNHX1
(AB038492), Atriplex gmelini; AtNHX1 (AF510074), AtSOS1 (AF256224), and AtSOS1 (AY062746), Arabidopsis
thaliana; NHE2 (AAD41635) and NHE5 (AAC98696), Homo sapiens; OsNHA1 (AY328087), Oryza sativa; TaSOS1
(AY326952), Triticum aestivum; PpSOS1 (AJ564258), Physcomitrella patens; PeNhaD1 (AJ561195), Populus
euphratica; NhaP (BAA31695), Pseudomonas aeruginosa; NHA1 (NP-013239), Saccharomyces cerevisiae; SOD2
(CAA77796), Schizosaccharomyces pombe; NhaA (P13738), Escherichia coli; and PeSOS1 (DQ517530), Populus
euphratica
Figure 3 - Hydrolytic activity of P-ATPase and PPiase and MDA concentration in roots of banana
genotypes grown for 7 days with or without 50 mol m-3 NaCl in the nutrient solution.
Significant difference between the means of the treatments according to F test (0,05%).
CHEN, T.H; MURATA, N. Glycinebetaine protects GIRI, J. Glycinebetaine and abiotic stress tolerance
plants against abiotic stress: mechanisms and in plants. Plant Signaling & Behavior, Austin, v.6,
biotechnological applications. Plant, Cell and p.1746-51, 2011.
Environment, Oxford, v.34, p.1–20, 2011. )
GOMES, E.W.F.; WILLADINO, L.; MARTINS,
FAÇANHA, A.R.; DE MÉIS, L. Reversibility of L.S.S.; SILVA, S.O.; CAMARA, T.R.; MEUNIER,
H+-ATPase and H+-pyrophosphatase in tonoplast I.M.J. Diplóides (AA) de bananeira submetidos ao
vesicles from maize coleoptiles and seeds. Plant estresse salino. Pesquisa Agropecuaria Brasileira,
Physiology, Washington, v.116, p.1487-95, 1998. Barsília, DF, v.39, p.525-31, 2004.
GOMES, E.W.F.; WILLADINO, L.; MARTINS, PARDO, J.M. Biotechnology of water and
L.S.S.; CAMARA, T.R. Variedades de bananeira salinity stress tolerance. Current Opinion in
tratadas com água salinizada em fase inicial de Biotechnology, Oxford, v.21, p.185-96, 2010.
crescimento. Revista Brasileira de Engenharia
Agrícola e Ambiental, Campina Grande, v.9, p.31- SHI, H.; ISHITANI, M.; KIM, C.; ZHU, J.K. The
6, 2005. Arabidopsis thaliana salt tolerance gene SOS1
encodes a putative Na+/H+ antiporter. Proceedings
GUPTA, B.; HUANG, B. Mechanism of salinity
of the National Academy Sciences, Washington,
tolerance in plants: physiological, biochemical,
v.97, p.6896–901, 2000.
and molecular characterization. International
Journal of Genomics, New York, v.2014,
SHI, H.; QUINTERO, F.J.; PARDO, J.M.; ZHU, J.K.
p.18, 2014. Disponível em: <http://dx.doi.org/
The putative plasma membrane Na+/H+ antiporter
urn:doi:10.1155/2014/701596>
SOS1 controls long-distance Na+ transport in plants.
Plant Cell, Rockwille, v.14, p.465-77, 2002. )
KUMAR, S.; TAMURAM K.; NEI, M. MEGA3:
Integrated software for molecular evolutionary
SILVA, R.L.O.; MARTINS, L.S.S.; GOMES,
genetics analysis and sequence alignment. Briefings
E.W.F.; WILLADINO, L. Avaliação de diploides
Bioinformatics, London, v.5, p.150-163, 2004.
de bananeira (Musa spp.) quanto à tolerância a
salinidade. Revista Brasileira de Fruticultura,
KUMAR, G.; PURTY R.S.; SHARMA,
Jaboticabal, v.31, n.4, p.1084-91, 2009.
M.P.; SINGLA-PAREEK, S.L.; PAREEK, A.
Physiological responses among Brassica species
SUN, W.; XU, X.; ZHU, H.; LIU, A.; LIU, L.; LI,
under salinity stress show strong correlation with
J.; HUA, X. Comparative transcriptomic profiling
transcript abundance for SOS pathway-related
of a salt-tolerant wild tomato species and a salt-
genes. Journal of Plant Physiology, Stuttgart,
sensitive tomato cultivar. Plant Cell & Physiology,
v.166, p.507-520, 2009.
Kyoto, v.51, p.997-1006, 2010.
KUMAR, R.R.; YADAD, S.; SHRINIVAS, D.;
THOMPSON, J.D.; HIGGINS D.G.; GIBSON, T.J.
SRIVASTAVA, A.K.; SHITOLE, S.; NAIK, G.R.
Improving the sensitivity of progressive multiple
Transcriptome of pigeonpea roots under water deficit
alignment through sequence weighting, position-
analyzed by suppression subtractive hybridization.
specific gap penalties and weight matrix choice.
Journal of Agricutural Science and Technology,
Nucleic Acids Research, London, v.22, p.4673-80,
Tehran, v.17, n.5, p.1333-45, 2015.
1994.
LIANG, P.; PARDEE, A.B. Recent advances
UEDA, A.; SHI , W.; NAKAMURA, T. ; TAKABE,
in differential display. Current Opinion in
T. Analysis of salt-inducible genes in barley roots
Immunology, London, v.7, p.274–80, 1995.
by differential display. Journal of Plant Research,
v.115, p.119-130, 2002.
MITTLER, R. Abiotic stress, the field environment
and stress combination. Trends in Plant Science,
VITART, V.; BAXTER, I.; DOERNER, P.;
Cambridge, v.11, p.16-9, 2006.
HARPER, J.F. Evidence for a role in growth and
salt resistance of a plasma membrane H+ATPase in
MILLER, G.; SUZUKI, N.; CIFTCI-YILMAZ,
the root endodermis. Plant Journal, Oxford, v.27,
S.; MITTLER, R. Reactive oxygen species
p.191-201, 2004.
homeostasis and signaling during drought and
salinity stresses. Plant, Cell & Environment,
WILLADINO, L.; GOMES, E.W.F.; SILVA,
Oxford, v.33, p.453–67, 2009.
E.F.F.; MARTINS, L.S.S.; CAMARA, T.R. Efeito
do estresse salino em genótipos tetraplóides de
MUNNS, R.; TESTER, M. Mechanism of salinity
bananeira. Revista Brasileira de Engenharia
tolerance. Annual Review Plant Biology, Palo
Agrícola e Ambiental, Campina Grande, v.15,
Alto, v.59, p.651-81, 2010.
p.53-9, 2011.